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Last revised: Monday, February 7, 2000
Ch. 5 in Prescott et al, Microbiology, 4th Ed.Note: These notes are provided as a guide to topics the instructor hopes to cover during lecture. Actual coverage will always differ somewhat from what is printed here. These notes are not a substitute for the actual lecture!Copyright 2000. Thomas M. Terry
- Energy source
- if organic molecules (e.g. sugars, amino acids) = ORGANOTROPH
- if inorganic molecules (e.g. H2S, NH3) = LITHOTROPH
- if light = PHOTOTROPH
- Macronutrients: Cell requires ~ 30 elements. Needs 6 in large amounts (CHNOPS), some only in trace amts, some in between
- can be CO2 = AUTOTROPH
- can be organic molecules = HETEROTROPH
- Note: many bacteria can form all organic molecules from any of a wide variety of C-sources. E.g., Pseudomonas strains (soil organisms) can grow on hydrocarbons, variety of sugars, fats, polysaccharides, etc.
- Full description of microbe typically refers to both E- and C-source; lithotrophic autotroph (chemolithotroph); organotrophic heterotroph, etc. Will see many examples of these.
- Typical bacterium is 15% nitrogen (in proteins, nucleic acids, cell wall).
- Most bacteria obtain either amino groups (-NH2) or ammonia (NH3), or nitrate (NO3--).
- Some bacteria can fix atmospheric nitrogen (N2) to form ammonia (NH3)
- generally available as phosphate ion (PO4---) or organic molecules (e.g. RNA).
- Often limiting for growth.
- required for 2 amino acids and for some vitamins.
- Available as sulfate (SO4--) or sulfide (S--).
- K, Mg, Ca, Fe required in small but significant amts. Act as cofactors for many enzymes, cell structures (e.g., Mg++ required by ribosomes for protein synthesis).
- Na+ not required by many microbes (unlike animals, where nerve system requires Na+ and K+). Marine microbes require sodium.
- Trace elements: Co, Zn, Mo, Cu, Mn, Ni, W, Se -- usually required by a small number of enzymes.
- Varieties of transport proteins:
- Uniporter -- protein transports substrate from one side of membrane to other (Passive transport -- no energy required)
- Symporter -- protein carries two substances across membrane in same direction (e.g. proton flux is coupled with motion of substrate). Active transport -- requires energy from preexisting concentration gradient.
- Antiporter -- protein carries one substance in one direction, second substance in opposite direction. Active transport -- requires energy from preexisting concentration gradient.
- Specific examples of transport processes
- Group translocation -- substance is chemically altered while being transported.
- Example: phosphotransferase system used in sugar transport. Sugars are common energy sources, bacteria have evolved complex transport system to bring them across membrane whenever possible.
- In E. coli, 24 proteins involved, can transport a variety of sugars (at least 4 proteins needed for any given sugar).
- Mechanism involves alternate phosphorylation (adding Pi group) and dephosphorylation (removing Pi group) of series of carriers. Initial phosphate donor is high-energy molecule PEP, passes Pi group to Enzme I --> HPr protein Enzyme IIa Enzyme IIb Enzyme IIc. Only the very last of these proteins is located in membrane; others are all found in cytoplasm. Sugar (e.g., glucose) is carried into cell by Enzyme IIc, and at same time phosphate group is added to make glucose 6-P, hence group translocation (sugar is modified by added Pi group)
- Active Transport -- substance is imported without chemical change, and energy is expended.
- Energy may be used in form of ATP
- More commonly, cells use energy in form of proton gradient or ion gradiend such as sodium gradient. After proton gradient is established (to be discussed later in section on energetics), symport proteins allow substate to brought in along with entry of proton.
- Media must include source of C, N, P, S, 4 of the 6 major nutrients (CHNOPS), as well as micronutrients. These are usually present as trace contaminants in water, on glassware, or in chemicals used to make media.
- Media can be liquid or solid. Use for different purposes:
- Liquid media: easiest to prepare and use. Good for growing quantities of microbes needed for analysis or experiments. Unless inoculated with pure culture, cannot separate different organisms.
- Solid media: usually made by adding agar, a seaweed extract, to appropriate liquid. 1.5% agar is standard for plates. Agar melts at 80-90 deg. C, will remain liquid until temperature cools to 40-42 deg. C. Very few microbes can degrade agar, so it is normally not a source of C, and acts as inert gelling medium.
- Many types of culture media; see BBL manual and DIFCO manual for formlations
- Synthetic of Defined Media: usually relatively simple media, all components are known. Useful for photoautotrophs, also in some experimental situations where want to select mutants unable to use certain compounds, or for radioisotope labeling. Example: you want to select a microbe that can obtain all its nitrogen from atmospheric N2. You would prepare synthetic medium with sources of C, P, and S, but no N source. Organisms would be unable to grow unless they can fix nitrogen from air.
- Complex Media: composition of media not completely known. Often made from inexpensive organic materials such as slaughterhouse wastes (tryptic digests called tryptone, trypticase, etc.), soybeans, yeast wastes from brewing (rich source of vitamins), animal blood, etc. All our standard laboratory media in MCB 229 are complex media, such as Tryptone agar, TSA (trypticase soy agar), Nutrient agar, etc.
- Selective Media: media favors the growth of one or more microbes. Example: bile salts inhibit growth of most gram-positive bacteria and some gram-negative bacteria, but enteric bacteria adapted to life in animal gut can grow well. Include bile salts in some media such as EMB, MacConkey agar (will use later in this course) to select for enterics.
- Differential Media: media allows distinguishing between different bacteria that grow. Ex: MacConkey agar has color indicator that distinguishes presence of acid. Bacteria that ferment a particular sugar (e.g., glucose in culture media) will produce acid wastes on plates, turn pH indicator red. Bacteria that cannot ferment the same sugar will grow but not affect pH, so colonies remain white.
- Note that it is possible to design a medium that is both selective and differential.
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